Carbonic Anhydrase 3 is required for cardiac repair post myocardial infarction via Smad7-Smad2/3 signaling pathway

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-β1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7‐TGF-β/Smad2/3 signaling pathway.


Supplementary Figures and Tables
Figure S1.The expression levels of CAR3 in normal and infarcted hearts post-MI.A, Protein expression of CAR3 in adult mice tissue was shown.B, The temporal protein expression patterns of CAR3 and CAR2 were detected in the infarct area by Western blot analysis (n = 4-6 mice per group) and RT q-PCR (n = 4-5 mice per group).C, Corresponding statistics of CAR3 and CAR2 were shown.D, CAR3 level at different time points was determined by Western blot (n = 4 mice per group) and RT q-PCR (n = 6 mice per group) in the border area of MI-operated hearts.E, Expression of CAR3 at different time points was measured using Western blot (n = 4-5 mice per group) and RT q-PCR (n = 4-5 mice per group) in the remote area of MI-treated hearts.The data are shown as the means ± SEM and analyzed by Student's t-test.CAR3, carbonic anhydrase 3; MI, myocardial infarction; CAR2, carbonic anhydrase 2; RT q-PCR, real-time quantitative polymerase chain reaction.Table S1.Primer sequences.

Figure S2 .
Figure S2.The expression of CAR3 showed no significant change in NRCMs exposed to hypoxia.A, Western blot bands of CAR3 in cultured NRCMs, NRCFs, and HUVECs.B, NRCMs were treated by hypoxia for 3h or 6h.CAR3 expression was examined by Western blot analysis (n = 5-6 per group) and RT q-PCR (n = 6 per group).C, Corresponding statistic of CAR3 was shown.D, IF co-staining for cTnT with CAR3 and DAPI in NRCMs exposed to hypoxia for 6h (n = 5 per group, scale bar = 20 μm).E, CAR3 level was determined by Western blot analysis in NRCFs treated with hypoxia for 12h or 24h (n = 4 per group).F, Expression of CAR3 was measured using Western blot analysis (n = 4 per group) in ACFs exposed to hypoxia for 12h or 24h.G. Protein level of CAR2 was detected by Western blot in cultured NRCFs treated with TGF-β1 (n = 6 per group).H, Expression of CAR2 was measured using Western blot in cultured ACFs treated with TGF-β1 (n = 6 per group).The data are shown as the means ± SEM.The data shown in C and E-Η were analyzed by one-way ANOVA followed by Bonferroni post hoc test, and D was analyzed by Student's t-test.NRCMs, neonatal rat cardiomyocytes; NRCFs, neonatal rat cardiac fibroblasts; HUVECs, human umbilical vein endothelial cells; cTnT, cardiac troponins T; DAPI, 4'6-diamidino-2-phenylindole; TGF-β1, transforming growth factor-β1; ACFs, adult mouse cardiac fibroblasts.

Figure S3 .
Figure S3.Validation of Car3-dificient in mice.A, Schematic diagram of Car3 gene-targeting strategy.B, Expression of CAR3 was measured using Western blot in normal (n = 8 mice per group) and infarcted hearts 7d post-MI from WT and Car3-deficient mice.C, Quantitative results for CAR3 in Western blot and RT q-PCR (n = 6 mice per group) were measured.Data are presented as mean ± SEM, and analyzed by Student's t-test.

Figure S4 .
Figure S4.The expression of CAR3 in cultured ACFs isolated from WT or Car3-knockout mice.A, Expression of CAR3 was measured by Western blot in ACFs from WT and Car3-deficient mice (n = 8 per group).B, Corresponding statistic of CAR3 was shown.Data are presented as mean ± SEM, and analyzed by Student's t-test.ACFs, adult mouse cardiac fibroblasts.

Figure S5 .
Figure S5.CAR3 deficiency exerted no significant effects on the expression of TGFβR1 or TGFβR2.A, Expressions of TGFβR1 was determined using Western blot (n = 4 per group) and RT q-PCR (n = 4 per group) in the infarct area of cardiac tissue post-MI.B, Expressions of TGFβR2 was measured by Western blot (n = 4 per group) and RT q-PCR (n = 4 per group) in the infarcted hearts after MI. Results were normalized against α-tubulin and converted to fold induction relative to control-treated group.C, TGFβR1 level was measured by Western blot (n = 4 per group) and RT q-PCR (n = 4 per group) in TGF-β1-treated ACFs.D, TGFβR2 level was determined using Western blot (n = 4 per group) and RT q-PCR

Figure S6 .
Figure S6.The expression of p300 in cultured ACFs and hearts from WT mice treated with DMSO or C646.A, Expressions of p300 was determined using Western blot (n = 4 independent experiments per group).B, Corresponding statistic of p300 was shown.C, Protein level of p300 in hearts from WT mice pretreated with DMSO or C646.D, Corresponding statistic of p300 was shown.Data are presented as mean ± SEM, and analyzed by Student's t-test.DMSO, Dimethyl sulfoxide.

Figure S7 .
Figure S7.The expression of CAR3 in cultured ACFs transduced with Adv-GFP or Adv-Car3 to overexpress CAR3 for 48 hours.A, Protein level of CAR3 was measured by Western blot (n = 6 independent experiments per group).B, Transcription level of Car3 was determined using RT q-PCR (n = 6 independent experiments per group).Data are presented as mean ± SEM, and analyzed by Student's t-test.GFP, green fluorescent protein.